Aldoheptose Biosynthesis. E. coli K-12 aldoheptose mutants, carrying the cysE-pyrE linked mutation rfaD or rfa-2, were previously isolated and genetically characterized. The rfaD phenotype includes increased permeability to a large number of hydrophobic antibiotics, in addition to altered LPS synthesis. The rfaD gene initially identified in the Clarke- Carbon Genomic Bank was cloned. The precise location of the rfaD gene on a 1.3-kilobase SspI-HpaI fragment has been determined. The rfaD gene and the flanking regions have been completely sequenced, and the coding and regulatory regions have been defined. The location of the rfaD gene on the physical map of the Escherichia coli chromosome has been resolved. RfaD plasmids express in vivo and in vitro a protein with the molecular weight of 37,000. The protein, ADP-L-glycero-D-mannoheptose-6-epimerase, has been purified to homogeneity and partially characterized. N-Terminal analysis of purified ADP-D-glycero-D-mannoheptose-6-epimerase confirms the first 34 amino acid sequence deduced from the nucleotide sequence of the rfaD gene coding region. The rfaD protein contains the fingerprint sequence of the ADP-binding beta-alpha-beta-fold of NAD-binding proteins, near its N- terminus. The rfaD gene studies have been extended to include the clinical pathogen Pseudomonas aeruginosa. The second cysE-pyrE linked mutation, designated rfa-2, resulted in heptoseless LPS (chemotype Re) and increased permeability to hydrophobic and hydrophilic agents. We have cloned the rfa-2 gene, resolved its sequence and flanking nucleotides, as well as determined its physical location on the E. coli chromosome. The rfa-2 gene encodes a 36Kd protein. Interspecific complementation studies demonstrated that the E. coli K-12 rfa-2 gene complements the chemotype Re LPS mutant of S. typhimurium, designated rfaC.